Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock
- University of Fribourg, Switzerland
Figures
Figure 1
CDK5 intersects with PER2 and has diurnal activity in the SCN.
(A) Loss of Eap1, Gnd1, or Pho85 compromises growth of PER2-overproducing yeast cells. The yeast mutants eap1∆, gnd1∆, and pho85∆ were identified in a synthetic dosage lethal screen as detailed …
Figure 2 with 5 supplements
CDK5 affects the circadian clock.
(A) Wheel-running activity of mice (black bins) infected with AAV expressing scrambled control shRNA, or shCdk5, and an animal with a deletion in the Per2 gene (Per2Brdm1). The actograms are double …
Figure 2—figure supplement 1
Characterization of shRNA against Cdk5 Western blot using NIH 3T3 cell extracts transfected with different shRNAs against Cdk5.
All shRNAs were mapped to the Cdk5 sequence. The Western blot reveals that the shRNA D (nucleotides 462 to 490) showed the best silencing activity.
Figure 2—figure supplement 2
Additional activity plots of wild-type mice infected with AAV.
Figure 2—figure supplement 3
Activity counts per day.
shCdk5 (13878 ± 2877 counts/day, n = 6) and Per2Brdm1 mice (10598 ± 1856 counts/day, n = 4) in DD when compared with the control animals (23478 ± 1277 counts/day, n = 6) as well as in LD conditions: …
Figure 2—figure supplement 4
Activity counts in dark or light phase.
Dark: scramble (23276 ± 2817 counts/day, n = 6), shCdk5 (10399 ± 3764 counts/day, n = 6), Per2Brdm1 mice (10521 ± 2052 counts/day, n = 4). Light: scramble (1301 ± 223 counts/day, n = 6), shCdk5 …
Figure 2—figure supplement 5
Additional activity plots of Per2Brdm1 mice infected with AAV.
Wheel-running activity (black bins) of Per2Brdm1 mice infected with AAV expressing scrambled control shRNA (scr), or shRNA against Cdk5 (shCdk5). The actograms are double plotted displaying in one …
Figure 3 with 4 supplements
Immunohistochemistry in the SCN of control and shCdk5 silenced wild type and Per2Brdm1 mice.
(A) Representative sections of the SCN region after injection of AAVs carrying either scrambled shRNA, or shCdk5. Slices were stained with DAPI (blue), or anti-GFP (green) and anti-CDK5 (red) …
Figure 3—figure supplement 1
Lower magnification of SCN sections stained for CDK5.
Representative brain sections of normal mice containing the SCN region after injection of AAVs carrying either scrambled shRNA or shCdk5. GFP was used as a marker to illustrate the infected region …
Figure 3—figure supplement 2
Quantification of CDK5 signal.
Higher magnification of representative sections of the SCN after AAVs carrying either scrambled shRNA (left column) or shCdk5 (right column). CDK5 if significantly down regulated in brain infected …
Figure 3—figure supplement 3
Lower magnification of SCN sections stained for PER2.
Representative brain sections of normal mice containing the SCN region after injection of AAVs carrying either scrambled shRNA or shCdk5. GFP was used as a marker to illustrate the infected region …
Figure 3—figure supplement 4
Quantification of PER2 signal.
Higher magnification of representative sections of the SCN after AAVs carrying either scrambled shRNA (left column) or shCdk5 (middle column). CDK5 is significantly down regulated in brain infected …
Figure 4 with 5 supplements
PER2 interacts with CDK5 in a temporal fashion in the cytoplasm.
(A) Overexpression of PER2 and CDK5-HA in NIH 3T3 cells and subsequent immunoprecipitation (IP) using an anti-CDK5 antibody. The left panel shows 5% of the input and the right panel co-precipitation …
Figure 4—figure supplement 1
PER2-CDK5 interaction in HEK cells.
Overexpression of PER2 and CDK5 in HEK 293 cells and subsequent immunoprecipitation (IP) using an anti-CDK5 antibody. The left panel shows the input and the right panel co-precipitation of PER2 with …
Figure 4—figure supplement 2
PER2-CDK5 interaction at different salt concentrations.
Immunoprecipitation (IP) of PER2 and CDK5 from total mouse brain extract collected at ZT12. Left panel shows the input. The middle and right panels depict co-immunoprecipitation of PER2 and CDK5 at …
Figure 4—figure supplement 3
Co-localization of PER2 and CDK5 in SCN tissue.
Temporal profile of the PER2-CDK5 interaction observed by immunofluorescence at ZT0 and ZT12. SCN slices, obtained from mice perfused at ZT 0 and ZT 12, were stained with anti-PER2 antibody (green) …
Figure 4—figure supplement 4
Deletion of PAS domains had no influence on PER2-CDK5 interaction.
NIH 3T3 cells were transfected with vectors carrying PER2-V5, ΔPasA-PER2-V5, or ΔPasB-PER2-V5, and subsequently, immunoprecipitation (IP) using an anti-CDK5 antibody was performed. The results …
Figure 4—figure supplement 5
Scheme of PER2 fragments used for the pull-down assy.
Figure 5 with 6 supplements
CDK5 phosphorylates PER2 at S394.
(A) An in vitro kinase assay was performed using recombinant CDK5/p35 and either GST-PER2 1–576 or GST-PER2 577–1256 as substrate. The samples were subjected to 10% SDS page (Coomassie, left panel) …
Figure 5—figure supplement 1
Scheme of PER2 fragments used for the in vitro kinase assay.
The fragment 1–576 covers the sites that might be phosphorylated by CDK5 on the basis of the conserved consensus (S/T)PX(K/H/R).
Figure 5—figure supplement 2
Additional controls for in vitro kinase assay.
An in vitro kinase assay performed in presence of recombinant CDK5/p35 and using as substrate the GST-PER2 1–576.
Figure 5—figure supplement 3
Testing specificity of the in vitro kinase assay.
The reactions were treated either with LiCl (inhibitor of GSK3β kinase activity) or roscovitine (inhibitor of CDK5 kinase activity) in order to highlight the specificity of the PER2 phosphorylation …
Figure 5—figure supplement 4
Characterization of antisera against P-S394-PER2.
Different antisera against the PER2 peptide sequence FDY{pSer}PIRFRTRNGEC were tested by in vitro kinase assay using recombinant GST-PER2 1–576 (in presence or absence of CDK5/p35) followed by WB. …
Figure 5—figure supplement 5
Characterization of hybridomas against P-S394-PER2.
Different hybridomas producing antibodies against the PER2 peptide sequence FDY{pSer}PIRFRTRNGEC were tested by in vitro kinase assay using recombinant GST PER2 1–576 (in presence or absence of …
Figure 5—figure supplement 6
Validation of anti-P-S394-PER2 antibody.
Total protein extracts were obtained from wild-type, Per2Brdm1 and Per2-/- mouse brains at ZT12. Western blot was performed in order to validate the specificity of the antibody against the …
Figure 6 with 4 supplements
CDK5 affects PER2 stability and nuclear localization.
(A) Western blot of NIH 3T3 cell extracts with and without roscovitine treatment. When roscovitine inhibited CDK5, less PER2 protein was detected in cell extracts. The bar diagram below shows values …
Figure 6—figure supplement 1
Characterization of Cdk5 ko cell morphology.
NIH 3T3 and CRISPR/Cas9 Cdk5-deficient cells were photographed using a bright light microscope (100 x). A clear difference in shape and thickness between the two cell lines could be observed. …
Figure 6—figure supplement 2
Selection for absence of Cdk5 mRNA.
PCR to detect the mutation of the genomic Cdk5 DNA sequence was performed on different putative knock-out clones. Among these, clones 3 and 5 showed the Cdk5 PCR product, demonstrating that showed …
Figure 6—figure supplement 3
Selection for absence of CDK5 protein.
Total protein extracts were obtained from clone 1, 10 and WT NIH 3T3. Western blot was performed in order to verify which clone no longer expressed CDK5. Clone number 10 was confirmed to be a …
Figure 6—figure supplement 4
Additional examples of cellular localization of PER2.
Additional examples of immunofluorescence of PER2 (red) at ZT12 in mouse SCN sections after infection with AAV (green) expressing scrambled shRNA (left column of panels), or shCdk5 (right column of …
Figure 7
Model illustrating the regulation of PER2 by CDK5.
The upper row illustrates phosphorylation of PER2 at S394 by CDK5 that subsequently favors interaction with CRY1 and leads to transport into the nucleus, where the PER2/CRY1 complex inhibits …
Figure 8
Workflow of the in vitro kinase assay.
Workflow of the in vitro kinase assay performed using immunoprecipitated CDK5 from SCN protein extracts is schematized here. Seven mice were sacrificed, SCN tissues were isolated and pooled together …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Per2Brdm1 | Jackson Laboratory | Stock #: 003819 | PMID: 10408444 |
| Genetic reagent (M. musculus) | B6;129P2-Per2tm1Ual/Biat | European mouse mutant archive | Strain ID EM:10599 | PMID: 26838474 |
| Cell line (M. musculus) | NIH3T3 | ATCC | Cat. #: ATCCRCRL-1658Tm | Immortalized Mouse fibroblast cells |
| Cell line (M. musculus) | NIH3T3 CRISPR/Cas9 Cdk5 KO | Origene | Cat. #: KN303042 | Immortalized Mouse fibroblast cells.7 ug/ml of puromycin are required for cells propagation |
| Cell line (Human) | HEK | ATCC | Immortalized Kidney fibroblast cells | |
| Transfected construct (M. musculus) | Sh RNA CDK5 plasmids | Origene | Cat. #: TL515615 A/B/C/D | |
| Transfected construct (M. musculus) | Sh RNA scramble | Origene | Cat. #: TR30021 | |
| Antibody | anti-PER2-1 (Rabbit polyclonal) | Alpha Diagnostic Lot # 869900A1.2-L | Cat. #: PER21-A RRID: AB_2236875: | 1:200 (IF) 1:50 (IP) 1:500/1:1000 (WB) |
| Antibody | anti-Cdk5 clone 2H6 (Mouse monoclonal) | Origene Lot # A001 | Cat. #: CF500397 RRID: AB_229166 | 1:20 (IF) 1:50 (IP) 1:500/1:1000 (WB) |
| Antibody | anti-GFP (Rabbit polyclonal) | Abcam | Cat. #: ab6556 RRID: AB_305564 | 1:500 (IF) |
| Antibody | anti-rabbit IgG (H+L) (Donkey polyclonal) | Alexa Fluor 488 Lot # 132876 | Cat. #: 711-545-152 RRID: AB_2313584 | 1:500 (IF) |
| Antibody | anti-mouse IgG (H+L) (Donkey polyclonal) | Alexa Fluor 647 Lot # 131725 | Cat. #: 715-605-150 RRID: AB_2340862 | 1:500 (IF) |
| Antibody | anti-rabbit IgG (H+L) (Donkey polyclonal) | Alexa Fluor 647 Lot # 136317 | Cat. #: 711-602-152 | 1:500 (IF) |
| Antibody | anti-HA (Mouse monoclonal) | Roche | Cat. #: 11583816001 RRID: AB_2532070 | 1:1000 (WB) |
| Antibody | anti-GST (Mouse monoclonal) | Sigma | Cat. #: G1160 RRID: AB_259845 | 1:1000 (WB) |
| Antibody | PER2 Phosphor Serine 133 (mouse monoclonal) | GenScript Company | Provided by the corresponding author | WB: 1:200 |
| Other | DAPI | Termofisher | Cat. #: D3571 RRID: AB_2307445 | (1 µg/mL) |
| Recombinant DNA reagent | Supplemental Table II | Complete list provided in the paper | ||
| Commercial assay or kit | pCR2.1-TOPO cloning | Thermofisher | Cat. #: K4500-01 | |
| Commercial assay or kit | QuikChange Site-Directed Mutagenesis Kit | Agilent | Cat. #: 200518 | |
| Chemical compound | Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K) | Polyscience Europe | Cat. #: 23966–1 | |
| Chemical compound | Roscovitine | Merk | Cat. #: R7772-1MG | |
| Chemical compound | Protein Agarose Beads | Roche | Cat. # 11 719 408 001 | |
| Chemical compound | cOmplete, EDTA-free Protease Inhibitor Cocktail | Merk | Cat. # 11873580001 | |
| Chemical compound | Isopropyl β-D-1-thiogalactopyranosid | Sigma-Aldrich | Cat. # 367-93-1 | |
| Chemical compound | L-Glutathione reduced | Merk | Cat. # 70-18-8 | |
| Chemical compound | Cycloheximide | Merk | Cat. # | |
| Chemical compound | Epoxomicin | Sigma-Aldrich | Cat. # | |
| Peptide, recombinant protein | Cdk5/p35 Protein, active, 10 µg | Millipore | Cat. # 14–477 | |
| Peptide, recombinant protein | Histone H1 | Sigma-Aldrich | Cat. # H1917-100UG | |
| Software | Prism | GraphPad | Version 8.2.0 | |
| Software | ImageJ | ImageJ | Version 1.49 RRID: SCR_00370 | |
| Software | ClockLab | Actimetrics | Acquistion version: 3.208 Analysis version: 6.0.36 RRID: SCR_0114309 | |
| Software | Leica application Suite Advanced Fluorescence | Leica | Version 2.7.3.9723 |
Additional files
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Supplementary file 1
Phosphorylation sites of GST-Per2 (1-576) detected by mass spectrometry.
The serine at position 394 stands out as the best localized phosphorylation site within a CDK5 consensus motif with a high peptide score (highlighted in yellow). The colored diagram shows the structural elements of PER2 (1–576) with the S394 phosphorylation site indicated. PEP: posterior error probability; Loc. Prob.; localization probability.
- https://cdn.elifesciences.org/articles/50925/elife-50925-supp1-v2.xlsx
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Supplementary file 2
Plasmids.
- https://cdn.elifesciences.org/articles/50925/elife-50925-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50925/elife-50925-transrepform-v2.docx
